Isolation And Phylogenetic Analysis Of Associated Bacteria From The Ciona Savignyi Collected From The Jiaozhou Bay And Taxonomic Analysis Of Two Marine Bacteria

Located at the warm-temperate district in China,the Jiaozhoui Bay contains rich natural resources and biological resources.Sea squirt is rich in associated bacteria,and great quantities of natural products with the novel structure and significant activity.Therefore,isolating associated bacteria from sea squirts will be a way to use the marine microorganism resources.In this study,associated bacteria isolated from sea squirt(Ciona savigny)collected from the Tsingtao Port,Jiaozhou Bay were cultured by using conventional culture-dependent method and phylogentic analysis based on the 16 S rRNA gene sequences comparisons,and the reults provided basic theory support for the development and utilization of marine microorganism.Meanwhile,two novel bacteria were studied by polyphasic approach in this study.There are seventeen culturable bacteria isolated from Ciona savigny by conventional culture-dependent method and neighbor-joining phylogentic analysis based on the 16 S rRNA gene sequences,and they have different colonies.The seventeen strains mainly belong to three phyla(Proteobacteria,Bacteroidetes and Actinobacteria).Twelve bacteria(accounted for 70.6% of total isolated stains)belong to Proteobacteria,and mainly belonging to Alphaproteobacteria and Gammaproteobacteria.Fermentation was carried out with isolated bacteria using four different mediums.The antibacterial activity of fermentation product was tested using five strains including Enterococcus faecium,Enterococcus faecalis,Staphylococcus aureus,Escherichia coli and Salmonclla typhimurium by agar diffusion method.The results showed that the strains could produce bioactive substance inhibiting E.faecium,E.faecalis and S.aureus were much more than E.coli and S.typhimurium;Marine ISP2 medium was the most likely to produce antimicrobial substances;The same strain still had difference in producing bioactive substance in different mediums;Fermentation products of strain H-7,H-8 and H-9 have greater inhibitory effects on E.faecium,E.faecalis and S.aureus than that on E.coli and S.typhimurium.Strain H-12 Twas isolated from a sea squirt(Ciona savigny)collected from the Tsingtao Port,Jiaozhou Bay by using 2216 E culture media.The 16 S rRNA gene sequence alignment results showed 95.3% similarity to that of Amylibacter marinus 2-3T.It was Gram-negative,non-motile,rod-shaped,approximately 0.6-1.1 ?m in width and 1.5-2.2 ?m in length.The colonies on MA were smooth,convex,opaque,light yellow,circular colonies after incubation for 4 days at 25 °C.Optimal growth of the strain was observed to be at 25-30 °C,pH 7.0-8.0 and with 3.0%-4.0%(w/v)NaCl;Growth did not occur under anaerobic conditions on MA.There were positive results in catalase,oxidase,hydrolysis of Tween 20 and 60.The DNA G+C content in this strain is 53.7 mol%.The predominant ubiquinone was Q-10.The predominant fatty acids were C18 : 1?7c,C18 : 1?7c 11-CH3 and C16 : 0.The major polar lipids were phosphatidylglycerol,phosphatidylcholine,phosphatidylethanolamine,one unidentified aminolipid,two unidentified phospholipid and one unidentified lipid.On the basis of these phenotypic,phylogenetic,chemotaxonomic data,strain H-12 T was believed to represent a novel species within the genus Amylibacter,for which the name Amylibacter cionae sp.nov.was proposed.The strain was H-12T(=KCTC 52581T=CGMCC 1.15880T).Strain T17 Twas isolated from marine sediment collected from the Tsingtao Port,Jiaozhou Bay by using 2216 E culture media.The 16 S rRNA gene sequence alignment results showed 96.0% similarity to that of Marinobacter lacisalsi LMG 24237 T.It was Gram-negative,rod-shaped and motile with one polar fiagella,approximately 0.6-1.2 ?m in width and 1.3-2.2 ?m in length.The colonies on MA were smooth,convex,opaque,cream white,circular colonies after incubation for 3 days at 35 °C.The colonies on MH were smooth,convex,opaque,light yellow,circular colonies.Optimal growth of the strain was observed to be at 25-30 °C,pH 7.0-8.0,and with3.0%-4.0%(w/v)NaCl.Growth does not occur under anaerobic conditions on MA.There were positive results in catalase,oxidase,amylase,hydrolysis of Tween 20,60,80 and H2 S production from cysteine.The DNA G+C content in the strain was 63.0 mol%.The predominant ubiquinone was Q-9.The predominant fatty acids were C12 : 0,C16 : 0,C16 : 0 10-CH3 and Sum feature 2*.On the basis of these phenotypic,phylogenetic,chemotaxonomic data,strain T17 T was believed to represent a novel species within the genus Marinobacter,for which the name Marinobacter bohaiensis sp.nov.was proposed.The strain was T17T(=KCTC 52710 T =MCCC 1K03282T).